Content
The purpose of this analytical report (2Ome_profile) is to evaluate
differences in rRNA 2’-O-methylation (2’Ome) levels between conditions
using C-score values computed across 112 annotated rRNA
sites. The objectives are:
- to provide a descriptive overview
of methylation profiles by condition prior to formal testing;
- to
determine whether methylation levels at individual sites differ
significantly between experimental conditions;
- to perform pairwise
comparisons between conditions using independent statistical
tests.
Project summary
This 2’Ome_diff report is generated from the analysis of the project
name, which is loaded into the RiboClass object
ribo_adj_annot_comp1. The dataset used for the
downstream analyses corresponds to a cohort of 12
biological samples, as part of the full dataset. This project was
aligned on GRCh38 (gencode annotation V46).
. Each
sample is annotated with associated metadata describing its biological
and technical context.
These metadata are essential for
interpreting the observed difference in 2’Ome level and for identifying
potential technical biases, such as batch effects, or biological
variations.
The following metadata are used in the project
name:
Method: RNA 2’Ome
interpretation and annotation
The 2’O-methylation (2’Ome) dataset consists of C-score
values measured at 112 annotated rRNA sites
for each biological sample. The C-score represents the level of 2’Ome of
a given site in a specific sample, ranging from 0 (unmethylated) to 1
(fully methylated). Intermediate values reflect heterogenous methylation
(i.e., heterogeneous methylation states across ribosomes within the same
cell or across the cells of the sample). The method used to compute the
C-score is detailed in the QC report (section 1.3 “Method”) and to
extract the sites of interest in the 2’Ome_profile report (section 1.3
“Method: RNA 2’Ome interpretation and annotationrRNA”). These C-scores
form the basis for all subsequent analyses, including global
profiling and differential analysis.
Here is a table, which summarizes the annotated rRNA 2’Ome sites used in
the downstream analyses where:
- “rna” column
indicates the RNA molecule on which the 2’Ome site is located
-
“rnapos” column indicates the position of the rRNA
2’Ome site
- “site” column indicates the name of
the rRNA 2’Ome site by combining the RNA molecule on which it is located
with the specific site identifier
Design of
comparisons
Comparisons were designed to evaluate the differential levels of
2’Ome at each of the 112 annotated sites between
biological conditions, as defined by the comp1 metadata
field in the name project dataset. Group comparisons
are performed when the dataset includes at least 2 conditions.
(1) Multiple comparisons are performed when the dataset includes 3 or
more conditions. In this case, all groups are considered together in
each comparison, enabling the identification of sites with significant
differences in 2’Ome levels in at least one group compared to the
others.
(2) Pairwise comparisons are performed when the dataset
includes 2 conditions. In this case, each comparison involves a case
group and a control group, enabling the identification of sites with
significant differences in 2’Ome levels:
| - cond2 vs. cond1:
6 samples in the cond2 (case) group
vs. 6 samples in the cond1 (control)
group |
These comparisons were designed to detect significant differences in
site-specific 2’Ome levels between the control condition and each test
condition. Both parametric and non-parametric statistical tests were
applied independently to each site within each comparison, and adjusted
p-values were computed to avoid bias due to the repetition of
statistical tests (see section 3 for details).
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